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Kazuyoshi Ukena

Publications and source records attributed to Kazuyoshi Ukena.

35 records · Page 2Linked to original sources

Organizing actions of neurosteroids in the Purkinje neuron.

It is becoming clear that steroids can be synthesized de novo by the brain of vertebrates. Such steroids synthesized de novo in the brain, as well as other areas of the nervous system, are called neurosteroids. To understand neurosteroid actions in the brain, we need data on the specific biosynthesis in particular sites of the brain at particular times. Therefore our studies for this exciting area of neuroscience research have focused on the biosynthesis and action of neurosteroids in the identified neurosteroidogenic cells underlying important brain functions. We have demonstrated that the Purkinje cell, a typical cerebellar neuron, is a major site for neurosteroid formation in the brain. This neuron actively synthesizes progesterone and estradiol de novo from cholesterol only during neonatal life, when cerebellar cortical formation occurs dramatically. This is the first observation of neuronal neurosteroidogenesis in the brain. Subsequently the actions of progesterone and estradiol during cerebellar development have become clear by a series of our studies using an excellent Purkinje cellular model. These neurosteroids promote dendritic growth, spinogenesis and synaptogenesis via each receptor in the Purkinje cell. Here we summarize the advances made in our understanding of organizing actions of neurosteroids in the Purkinje cell, an important brain neuron.

Animals↗

Photoperiodic response of serotonin- and galanin-immunoreactive neurons of the paraventricular organ and infundibular nucleus in Japanese quail, Coturnix coturnix japonica.

We investigated the photoperiodic response of serotonin- and galanin (GA)- immunoreactive (ir) cells in the paraventricular organ (PVO) and infundibular nucleus (IF) of the Japanese quail and the interaction of these cells with gonadotropin-releasing hormone (GnRH)-ir neurons in the hypothalamus. Serotonin-ir cells were located in series from the PVO to the IF, and were connected with each other. The number of serotonin-ir cells differed significantly between light and dark phases on the short days (SD), but did not differ between light and dark phases on long days (LD). GA-ir cells were also found in the PVO and IF. The number of GA-ir cells under SD conditions was significantly greater than under LD conditions but did not change diurnally. Both serotonin-ir and GA-ir fibers ran along the GnRH-ir cells in the nucleus commissurae pallii. Serotonin-ir and GA-ir fibers were connected with the GnRH-ir fibers in the external layer of the median eminence (ME). We confirmed that GA-ir fibers were closely associated with serotonin-ir neurons in the PVO and IF. GA-ir neurons have at least 2 routes of regulating GnRH neurons directly, and indirectly via the serotonin-ir cells in the PVO and IF.

Animals↗

Dendritic growth and spine formation in response to estrogen in the developing Purkinje cell.

Neurosteroids are synthesized de novo in the brain, and the cerebellar Purkinje cell is a major site for neurosteroid formation. We have demonstrated that the Purkinje cell possesses intranuclear receptor for progesterone and actively produces progesterone de novo from cholesterol only during rat neonatal life, when cerebellar cortical formation occurs dramatically. We have further demonstrated that progesterone promotes dendritic growth, spinogenesis, and synaptogenesis via its receptor in this neuron in the neonate. On the other hand, estrogen may also play an important role in the process of cerebellar cortical formation, because the neonatal rat Purkinje cell possesses estrogen receptor (ER)beta. However, estrogen formation in the neonatal cerebellum is still unclear. In this study, we therefore analyzed the biosynthesis and action of estrogen in Purkinje cells during neonatal life. RT-PCR-Southern and in situ hybridization analyses showed that Purkinje cells expressed the key enzyme of estrogen formation, cytochrome P450 aromatase, in neonatal rats. A specific enzyme immunoassay for estradiol further indicated that cerebellar estradiol concentrations in the neonate were significantly higher than those in the prepuberty and adult. Both in vitro and in vivo studies with newborn rats showed that estradiol promoted dose-dependent dendritic growth of Purkinje cells. Estradiol also increased the density of Purkinje dendritic spines. These effects were inhibited by the ER antagonist tamoxifen. These results suggest that estradiol in the developing Purkinje cell promotes dendritic growth and spinogenesis via ERbeta in this neuron. Estradiol as well as progesterone may contribute to the growth of Purkinje cells during the cerebellar cortical formation.

Animals↗

Neonatal expression of progesterone receptor isoforms in the cerebellar Purkinje cell in rats.

The cerebellar Purkinje cell (PC) is a typical site for neurosteroid formation. Progesterone synthesized de novo in developing PCs participates in the promotion of dendritic growth, spinogenesis and synaptogenesis in this neuron and such organizing actions may contribute to the formation of the cerebellar neuronal circuit during rat neonatal life. Progesterone receptors (PR) occur as two isoforms (PR-A and PR-B) derived from a single gene. To clarify the mode of organizing actions of progesterone, therefore, we examined the expression of these PR isoforms in the rat cerebellum during development. Western immunoblot analysis revealed that both PR isoforms were expressed highly in the cerebellum during neonatal life and the expression decreased thereafter. PR-like immunoreactivity was localized primarily in PCs in the neonatal cerebellum. Thus, progesterone may act directly on PCs via PR isoforms to promote its dendritic growth, spinogenesis and synaptogenesis.

Animals↗

Distribution of a novel avian gonadotropin-inhibitory hormone in the quail brain.

We recently identified a novel hypothalamic neuropeptide inhibiting gonadotropin release in the quail brain and termed it gonadotropin inhibitory hormone (GnIH). In this study, we investigated the localization and distribution of GnIH in both sexes of adult quails by immunohistochemistry with a specific antiserum against GnIH and in situ hybridization. Quantitative analysis demonstrated that the concentration of GnIH in the diencephalon was greater than that in the mesencephalon without sex difference. GnIH concentrations in the cerebrum and cerebellum were below the level of detectability. Clusters of GnIH-like immunoreactive (GnlH-ir) cell bodies were localized in the paraventricular nucleus (PVN) of the hypothalamus. There was no significant difference in the number of GnlH-ir cells in the PVN between males and females. By double immunostaining with antisera reacting with GnIH or avian posterior pituitary hormones (vasotocin and mesotocin), GnIH-ir cells were found to be parvocellular neurons in the ventral portion of PVN, which showed no immunoreaction with the antisera against vasotocin and mesotocin. In situ hybridization revealed the cellular localization of GnIH mRNA in the PVN. GnIH-ir nerve fibers were however widely distributed in the diencephalic and mesencephalic regions. Dense networks of immunoreactive fibers were found in the ventral paleostriatum, septal area, preoptic area, hypothalamus, and optic tectum. The most prominent fibers were seen in the median eminence of the hypothalamus and the dorsal motor nucleus of the vagus in the medulla oblongata. Thus, GnIH may participate not only in neuroendocrine functions, but also in behavioral and autonomic mechanisms.

Animals↗

Seasonal changes in neurosteroid concentrations in the amphibian brain and environmental factors regulating their changes.

Up to now the regulatory mechanisms, which govern the concentrations of neurosteroids in the brain, are unclear. Seasonal breeders may serve as excellent models to understand physiological changes in neurosteroid levels and their regulatory mechanisms. The present study first investigated immunohistochemically the localization of cytochrome p450 side-chain cleavage enzyme (p450scc) and 3beta-hydroxysteroid dehydrogenase/delta(5)-delta(4)-isomerase (3betaHSD) in the brain of the newt Cynops pyrrhogaster, a seasonally breeding amphibian. Both p450scc- and 3betaHSD-like immunoreactive cells were restricted to the preoptic area. Seasonal changes in neurosteroid concentrations were then examined using adult males. Pregnenolone concentrations in the brain showed marked changes during annual breeding cycle and a maximal level in August, independent of the plasma steroid levels which were all low throughout the year. Progesterone concentrations in the brain, which were lower than pregnenolone levels, also showed peaks in April and August. In contrast, the pregnenolone sulfate level was low and its change was less pronounced. To clarify environmental factors that induce seasonal changes in neurosteroid levels, adult males were further subjected to different photoperiods and ambient temperatures for 5 weeks. Both pregnenolone and progesterone concentrations in the brain were significantly higher in the long day (LD) group than in the short day (SD) group, whereas no significant effects of different ambient temperatures on neurosteroid levels were detected. These results suggest that the newt brain possesses steroidogenic enzymes p450scc and 3betaHSD and exhibits seasonal changes in the pregnenolone and progesterone concentrations. Photoperiod may be a more important environmental factor than temperature for the regulation of neurosteroid levels in the brain.

Amphibians↗

Biosynthesis and action of neurosteroids in the cerebellar Purkinje neuron.

The brain is considered to be a target site of peripheral steroid hormones. In contrast to this classical concept, new findings over the past decade have established that the brain itself also synthesizes steroids de novo from cholesterol through mechanisms at least partly independent of peripheral steroidogenic glands. Such steroids synthesized de novo in the brain, as well as other areas of the nervous system, are called neurosteroids. To understand neurosteroid actions in the brain, we need data on the specific synthesis in particular sites of the brain at particular times. Therefore, our studies for this exciting area of brain research have focused on the biosynthesis and action of neurosteroids in the identified neurosteroidogenic cells underlying important brain functions. We have demonstrated that the Purkinje cell, a typical cerebellar neuron, is a major site for neurosteroid formation in the brain. This is the first observation of neuronal neurosteroidogenesis in the brain. Subsequently, genomic and nongenomic actions of neurosteroids have become clear by a series of our studies using an excellent Purkinje cellular model. On the basis of these findings, we summarize the advances made in our understanding of biosynthesis and action of neurosteroids in the cerebellar Purkinje cell.

3-Hydroxysteroid Dehydrogenases↗

A novel aspect of the cerebellum: biosynthesis of neurosteroids in the Purkinje cell.

Peripheral steroid hormones act on brain tissues through intracellular receptor-mediated mechanisms to regulate several important brain neuronal functions. The brain is therefore considered to be a target site of steroid hormones. In contrast to this classical concept, new findings over the past decade have established that the brain itself also synthesizes steroids de novo from cholesterol through mechanisms at least partly independent of peripheral steroidogenic glands. Such steroids synthesized de novo in the brain, as well as other areas of the nervous system, are called neurosteroids. To analyze neurosteroid actions in the brain, we need data on the specific synthesis in particular sites of the brain at particular times. Such information is crucial to developing hypotheses predicting the potential roles of particular neurosteroids in the developing and adult brains. Thus our studies for this exciting area of brain research have focused on the biosynthesis of neurosteroids in the identified neurosteroidogenic cells underlying important brain functions. We have demonstrated that the Purkinje cell, a typical cerebellar neuron, is a major site for neurosteroid formation in the brain. This is the first observation of neuronal neurosteroidogenesis in the brain. Subsequently genomic and nongenomic actions of neurosteroids have been suggested by a series of our studies using an excellent Purkinje cellular model. Here we summarize the advances made in our understanding of biosynthesis of neurosteroids in the cerebellar Purkinje cell.

Animals↗

Novel neuropeptides related to frog growth hormone-releasing peptide: isolation, sequence, and functional analysis.

We previously identified in the bullfrog a novel hypothalamic RFamide peptide (SLKPAANLPLRF-NH(2)) that stimulated GH release in vitro and in vivo and therefore was designated frog GH-releasing peptide (fGRP). Molecular cloning of cDNA encoding the deduced fGRP precursor polypeptide further revealed that it encodes fGRP and its related peptides (fGRP-RP-1, -RP-2, and -RP-3). In this study immunoaffinity purification using the antibody against fGRP was therefore conducted to determine whether these three putative fGRP-RPs exist as mature endogenous ligands in the frog brain. The mass peaks of the isolated immunoreactive substances were detected at 535.78, 1034.14, and 1079.71 m/z ([M+2H](2+)), and their sequences, SIPNLPQRF-NH(2), YLSGKTKVQSMANLPQRF-NH(2), and AQYTNHFVHSLDTLPLRF-NH(2), were revealed by the fragmentation, showing mature forms encoded in the cDNA sequences of fGRP-RP-1, -RP-2, and -RP-3, respectively. All of these fGRP-RPs contained a C-terminal -LPXRF-NH(2) (X = L or Q) sequence, such as fGRP. This study further analyzed hypophysiotropic activities of the identified endogenous fGRP-RPs. Only fGRP-RP-2 stimulated, in a dose-related way, the release of PRL from cultured frog pituitary cells; its threshold concentration ranged from less than 10(-7) M. A similar stimulatory action of fGRP-RP-2 on GH release was evident. It was ascertained that fGRP-RP-2 was also effective in elevating the circulating GH and PRL levels when administered systemically. In contrast, fGRP-RPs did not have any appreciable effect on the release of gonadotropins. Thus, fGRP-RP-2 may act as a novel hypothalamic factor on the frog pituitary to stimulate the release of GH and PRL.

Amino Acid Sequence↗

Expression and localization of cytochrome P450(11beta,aldo) mRNA in the frog brain.

The present study is focused on biosynthesis of adrenal steroids in the frog brain. Employing RT-PCR method using total RNA from the adult Rana nigromaculata brain, we isolated a 419-bp fragment of cDNA encoding cytochrome P450(11beta,aldo), which catalyzes the final step of biosynthesis of the frog adrenal steroids, corticosterone and aldosterone. The deduced amino acid sequence of R. nigromaculata brain cytochrome P450(11beta,aldo) shared a high homology (88.8%) with that of R. catesbeiana adrenal cytochrome P450(11beta,aldo). Southern blot analysis of the RT-PCR product confirmed the P450(11beta,aldo) transcription in the frog brain without a clear-cut sex difference. Then, we analyzed the P450(11beta,aldo) mRNA expression in different brain regions of the adult frog by RT-PCR method. The P450(11beta,aldo) gene was transcribed in the telencephalon, diencephalon, midbrain, and cerebellum. The transcript level of the frog beta-actin gene was relatively constant in all the frog samples examined. In situ hybridization analysis showed that the P450(11beta,aldo) gene was transcribed abundantly in the cells throughout the frog brain, such as the pallium mediale in the telencephalon, the nucleus preopticus in the diencephalon, the stratum griseum superficiale tecti in the midbrain, and Purkinje cells in the cerebellum. These results taken together suggest that the frog brain synthesizes adrenal steroids, such as corticosterone and aldosterone.

Amino Acid Sequence↗

Androgen biosynthesis in the quail brain.

We have demonstrated that the quail brain possesses the cholesterol side-chain cleavage enzyme (cytochrome P450scc) and 3beta-hydroxysteroid dehydrogenase/delta(5)-delta(4)-isomerase (3beta-HSD) and produces pregnenolone, pregnenolone sulfate and progesterone from cholesterol. We have also demonstrated the expression of cytochrome P450 17alpha-hydroxylase/c17,20-lyase (P450(17alpha,lyase)) and the conversion of progesterone to 17alpha-hydroxyprogesterone in the same avian species. Therefore, the present study was conducted to investigate androgen biosynthesis from progesterone in the avian brain. Employing biochemical techniques combined with HPLC and TLC analyses, the conversion of progesterone to androstenedione, an androgen precursor, was found in quail brain. The present biochemical analysis further revealed the conversion of androstenedione to testosterone, indicating the presence of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) in the quail brain. The formation of testosterone from progesterone was also detected in the brain. Testosterone formation was more intense in the diencephalon, whereas the concentration of endogenous testosterone in the diencephalon was lower than those in other brain regions in castrated quails. However, the concentration of endogenous estradiol, a metabolite of testosterone by cytochrome P450arom, was highest in the diencephalon of castrated quails. These results suggest that testosterone biosynthesis occurs in the quail brain, in particular the diencephalon. Testosterone may subsequently be converted to estradiol.

17-Hydroxysteroid Dehydrogenases↗

Dendritic spine formation in response to progesterone synthesized de novo in the developing Purkinje cell in rats.

The cerebellar Purkinje cell (PC) is a typical site for neurosteroid formation. We have demonstrated that this neuron possesses intranuclear receptor for progesterone and actively synthesizes progesterone de novo from cholesterol only during rat neonatal life, when the formation of the cerebellar cortex occurs dramatically. In this study, we therefore analyzed the effect of progesterone on dendritic spine formation of the PC. In vitro studies using cerebellar slice cultures from newborn rats showed that progesterone increases the density of PC dendritic spines in a dose-dependent manner. This effect was blocked by the progesterone receptor antagonist, RU486. Furthermore, trilostane, a specific inhibitor of progesterone synthesis, inhibited the increase of spine density. These results suggest that progesterone can promote dendritic spine formation, and endogenous progesterone synthesized de novo in the developing PC may induce such an effect.

Animals↗

Isolation and characterization of a GnRH-like peptide from Octopus vulgaris.

Gonadotropin-releasing hormone (GnRH) is the key peptide in the hypothalamo-hypophysial-gonadal axis, the core of regulation of reproduction in vertebrates. In this study, an octopus peptide with structural features similar to vertebrate GnRHs was isolated from brains of Octopus vulgaris. This peptide showed luteinizing hormone-releasing activity in quail anterior pituitary cells. A cDNA encoding the precursor protein was cloned. The RT-PCR transcripts were expressed in the supraesophageal and subesophageal brains, peduncle complex, and optic gland. The presence of the peptide in the different brain region was confirmed with enzyme-linked immunosorbent assay and time-of-flight mass spectrometric analysis. Immunoreactive neuronal cell bodies and fibers were observed in the subpedunculate lobe that controls the optic-gland activity. Optic gland nerves and glandular cells in the optic gland were immunostained. The isolated peptide may be octopus GnRH that contributes to octopus reproduction not only as a neurohormone but also as an endocrine hormone.

Amino Acid Sequence↗

A novel rat hypothalamic RFamide-related peptide identified by immunoaffinity chromatography and mass spectrometry.

Recently, cDNAs encoding novel RFamide-related peptides (RFRPs) have been reported in the mammalian brains by a gene database search and the deduced RFRPs have been suggested to participate in neuroendocrine and pain mechanisms in the rat. Two peptides have been predicted to be encoded in the cDNA of rodent RFRPs. To assess precise functions of rodent RFRPs in the brain, in the present study we identified a naturally occurring RFRP in the rat hypothalamus by immunoaffinity purification combined with mass spectrometry (MS). The affinity chromatography showed that the rat hypothalamus contained RFRP-like immunoreactivity. The immunoreactive material was analyzed by a nanoflow electrospray ionization time-of-flight MS followed by tandem MS analysis. The mass peak corresponding to octadecapeptide was detected at 1010.54 m/z ([M+2H](2+)) and its sequence, ANMEAGTMSHFPSLPQRF-NH(2), was revealed by the fragmentation, showing a mature form encoded in the cDNA sequence of RFRPs. The identified endogenous RFRP will aid not only in defining its physiological roles but also facilitate the development of its agonists and antagonists in the rodent brain.

Amino Acid Sequence↗

Novel fish hypothalamic neuropeptide.

Recently, we identified novel avian and amphibian hypothalamic neuropeptides that inhibited gonadotropin release and stimulated growth hormone release. They were characterized by a similar structure including the C-terminal LPLRF-NH2 motif. To clarify that the expression of these novel hypothalamic neuropeptides is a conserved property in vertebrates, we characterized a cDNA encoding a similar novel peptide, having LPLRF-NH2 from the goldfish brain, by a combination of 3' and 5' rapid amplification of cDNA ends (RACE). The deduced peptide precursor consisted of 197 amino acid residues, encoding three putative peptide sequences that included -LPXRF (where X is L or Q) at their C-termini. Mass spectrometric analyses revealed that a tridecapeptide (SGTGLSATLPQRF-NH2) was derived from the precursor in the brain as an endogenous ligand. Southern blotting analysis of reverse-transcriptase-mediated PCR products demonstrated a specific expression of the goldfish peptide gene in the diencephalon. In situ hybridization revealed the cellular localization of goldfish peptide mRNA in the nucleus posterioris periventricularis in the hypothalamus. Immunoreactive cell bodies were also restricted to the the nucleus posterioris periventricularis and the nervus terminalis and immunoreactive fibers were distributed in several brain regions including the nucleus lateralis tuberis pars posterioris and pituitary. Thus, the goldfish hypothalamus expresses a novel neuropeptide containing the C-terminal -LPQRF-NH2 sequence, which may possess multiple regulatory functions and act, at least partly, on the pituitary to regulate pituitary hormone release.

Amino Acid Motifs↗

A novel amphibian hypothalamic neuropeptide: isolation, localization, and biological activity.

Neuropeptides similar to the molluscan cardioexcitatory Phe-Met-Arg-Phe-NH2 have been identified in several vertebrates and characterized by the RFa motif at their C terminus (RFa peptides). In this study, we sought to identify an amphibian hypothalamic RFa peptide that may regulate secretion of hormones by the anterior pituitary gland. An acid extract of bullfrog hypothalami was passed through C-18 reversed-phase cartridges, and then the retained material was subjected to HPLC, initially using a C-18 reversed-phase column. RFa immunoreactivity was measured in the eluted fractions by a dot immunoblot assay employing an antiserum raised against RFa. Immunoreactive fractions were subjected to further cation exchange and reversed-phase HPLC purification. The isolated peptide was a novel RFa peptide and shown to have the sequence Ser-Leu-Lys-Pro-Ala-Ala-Asn-Leu-Pro-Leu-Arg-Phe-NH2. The cell bodies and terminals containing this peptide were localized immunohistochemically in the suprachiasmatic nucleus and median eminence, respectively. This RFa peptide stimulated, in a dose-related way, the release of GH from cultured pituitary cells, its threshold concentration ranging between 10(-9) and 10(-8) M. This peptide did not have any appreciable effect on the secretion of PRL and gonadotropins. It was ascertained that the peptide was also effective in elevating the circulating GH level when administered systemically. Thus, the amphibian hypothalamus was revealed to contain a novel functional RFa peptide that stimulates GH release. This peptide was designated frog GH-releasing peptide.

Aging↗

A new member of the hypothalamic RF-amide peptide family, LPXRF-amide peptides: structure, localization, and function.

Recently, we identified a novel hypothalamic neuropeptide with a C-terminal LPLRF-amide sequence in the quail brain. This avian neuropeptide was shown to inhibit gonadotropin release from the cultured anterior pituitary. This peptide is the first hypothalamic peptide that inhibited gonadotropin release reported in vertebrates. We, therefore, termed it gonadotropin-inhibitory hormone (GnIH). After this finding, we found that GnIH-related peptides were present in the brains of other vertebrates, such as mammals, amphibians, and fish. These GnIH-related peptides possessed a LPXRF-amide (X=L or Q) motif at their C-termini in all investigated animals. Mass spectrometric analyses combined with immunoaffinity chromatography were powerful techniques for the identification of mature endogenous LPXRF-amide peptides. The identified LPXRF-amide peptides were found to be localized in the hypothalamus and brainstem areas, and to regulate pituitary hormone release. Subsequently, cDNAs that encode LPXRF-amide peptides were characterized in vertebrate brains. In this review, we summarize the identification, localization, and function of a new member of the hypothalamic RF-amide peptide family, LPXRF-amide peptides in vertebrates. Recent studies on the receptors for LPXRF-amide peptides will also be reviewed.

Amides↗